In eukaryotic organisms, there are commonly three types of RNA polymerases, that is, I to III. The polymerase III synthesizes low molecular weight RNA genes such as transfer RNA (hereinafter, referred to as “tRNA”) and small nuclear RNAs (hereinafter, referred to as “snRNA”. In the RNA synthesis by the polymerase III, the transcription start site is precisely determined, and the transcription is terminated by a sequence of five consecutive thymidines on DNA. In addition, at the end of the transcription, two or three adenyls are added. The polymerase III promoter includes two types of promoters, that is, a promoter with a TATA box, and a promoter without a TATA box. The polymerase III promoter with a TATA box is utilized for analysis of the gene function as described below.
In the post-genome sequence era, as a method for analyzing the gene function, an approach of reverse genetics such as RNA interference (hereinafter, referred to as “RNAi”) by which determination of the base sequence of genes leads to identification of its function has gained attention. The RNAi is a phenomenon in which a double-stranded RNA (hereinafter, referred to as “dsRNA”) constituted by a sense RNA that is homologous to a specific region of a gene whose function is desired to be inhibited and an anti-sense RNA interferes and destroys the homologous portion of mRNA that is a transcription product of the target gene.
For example, when a synthesized short chain interfering RNA (hereinafter, referred to as “siRNA”) constituted by dsRNA of less than 30 base pairs is introduced into a mammal cell, mRNA having a homologous sequence is degraded. As a result, expression of proteins to which the target mRNA is to be translated is significantly reduced. Similarly, it has been made evident that RNAi can be induced by introducing a DNA vector expressing sense chain and anti-sense chain RNA simultaneously, or a hairpin RNA (hereinafter, referred to as “hpRNA”) of invert repeat sequences, to a mammal cell. A method using the RNA polymerase III promoter at this time has been developed.
Nature Biotech (2002) 20: 446-448
Nature Biotech (2002) 19: 497-500
The RNA synthesis method employing RNA polymerase III promoters has made it possible to try to isolate a cell where RNAi is occurred constantly or to produce a transgenic (TG) mouse where RNAi is occurred for the purpose of analyzing the gene function.
Proc Natl Acad Sci USA. (2002) 99(8):5515-20
FEBS lett. (2002) 532 (1-2): 227-30
However, RNAi is a mechanism for suppressing gene expression, so that growth of a cell can be significantly affected, depending on the target gene whose expression is to be suppressed, and therefore it can be predicted that the cell where RNAi is occurred cannot be cloned or that a TG mouse that induces RNAi dies in the fetus stage. In order to prove a connection between suppressor genes and the abnormality of a phenotype more precisely, it is necessary to trigger RNAi in the development stage or only a specific tissue (organ) for analysis of the gene function.
In view of the above, it is expected to establish a method for inducing RNAi with the condition that RNAi can be induced in an arbitrary point of time, or only in a specific tissue (organ).
On the other hand, as a method for controlling so that a target gene becomes defective only under certain conditions or in a certain issue, a Cre-loxP system has been developed, and is used to produce, for example, knock-out mice or the like.
JP H1-112986A
This system utilizes the fact that Cre recombinase of a bacteriophage PI recognizes a loxP sequence of 34 bases, and DNA recombination is caused so that a loop-out occurs in a region between the loxP sequences, and this Cre-loxP system is known to function in mammal cells. In this system, when a configuration in which a stuffer flanked by the loxP sequences is arranged between a promoter and a target gene is used, in general, only the gene in the stuffer is expressed, and expression of the target gene is completely suppressed. However, Cre recombinase acts thereon, a homologous recombination occurs between the two loxP sequences. As a result, the stuffer gene is removed, and expression of the target gene is induced.
(Special issue of experimental medical science, The protocol series Experimental method for gene function inhibition, pp. 33 to 37, Tanabe, 2001)
Then, the inventors of the present invention tested to combine the RNAi method and the Cre-loxP system. However, when the target gene is a gene that is transcribed to a molecular inducing RNAi, in the Cre-loxP system in which a stuffer sequence flanked by loxP sequences is introduced between a promoter and a target gene, one of the loxP sequences remains, so that this is also transcribed together with the target gene and inserted in the RNAi molecule, so that the RNAi molecules results in having an inappropriate structure. As a result, the originally target RNAi failed to be occurred.